1.1 Field of the Invention
This invention relates to transformed host cells and vectors which comprise nucleic acid segments encoding genetically-engineered, recombinant Bacillus thuringiensis δ-endotoxins which are active against Coleopteran insects.
1.2 Description of the Related Art
Almost all field crops, plants, and commercial farming areas are susceptible to attack by one or more insect pests. Particularly problematic are Coleopteran and Lepidopteran pests. For example, vegetable and cole crops such as artichokes, kohirabi, arugula, leeks, asparagus, lentils, beans, lettuce (e.g., head, leaf, romaine), beets, bok choy, malanga, broccoli, melons (e.g., muskmelon, watermelon, crenshaw, honeydew, cantaloupe), brussels sprouts, cabbage, cardoni, carrots, napa, cauliflower, okra, onions, celery, parsley, chick peas, parsnips, chicory, peas, chinese cabbage, peppers, collards, potatoes, cucumber, pumpkins, cucurbits, radishes, dry bulb onions, rutabaga, eggplant, salsify, escarole, shallots, endive, soybean, garlic, spinach, green onions, squash, greens, sugar beets, sweet potatoes, turnip, swiss chard, horseradish, tomatoes, kale, turnips, and a variety of spices are sensitive to infestation by one or more of the following insect pests: alfalfa looper, armyworm, beet armyworm, artichoke plume moth, cabbage budworm, cabbage looper, cabbage webworm, corn earworm, celery leafeater, cross-striped cabbageworm, european corn borer, diamondback moth, green cloverworm, imported cabbageworm, melonworm, omnivorous leafroller, pickleworm, rindworm complex, saltmarsh caterpillar, soybean looper, tobacco budworm, tomato fruitworm, tomato hornworm, tomato pinworm, velvetbean caterpillar, and yellowstriped armyworm. Likewise, pasture and hay crops such as alfalfa, pasture grasses and silage are often attacked by such pests as armyworm, beef armyworm, alfalfa caterpillar, European skipper, a variety of loopers and webworms, as well as yellowstriped armyworms.
Field crops such as canola/rape seed, evening primrose, meadow foam, corn (field, sweet, popcorn), cotton, hops, jojoba, peanuts, rice, safflower, small grains (barley, oats, rye, wheat, etc.), sorghum, soybeans, sunflowers, and tobacco are often targets for infestation by insects including armyworm, asian and other corn borers, banded sunflower moth, beet armyworm, bollworm, cabbage looper, corn rootworm (including southern and western varieties), cotton leaf perforator, diamondback moth, european corn borer, green cloverworm, headmoth, headworm, imported cabbageworm, loopers (including Anacamptodes spp.), obliquebanded leafroller, omnivorous leaftier, podworm, podworm, saltmarsh caterpillar, southwestern corn borer, soybean looper, spotted cutworm, sunflower moth, tobacco budworm, tobacco hornworm, velvetbean caterpillar,
Bedding plants, flowers, ornamentals, vegetables and container stock are frequently fed upon by a host of insect pests such as armyworm, azalea moth, beet armyworm, diamondback moth, ello moth (hornworm), Florida fern caterpillar, Io moth, loopers, oleander moth, omnivorous leafroller, omnivorous looper, and tobacco budworm.
Forests, fruit, ornamental, and nut-bearing trees, as well as shrubs and other nursery stock are often susceptible to attack from diverse insects such as bagworm, blackheaded budworm, browntail moth, california oakworm, douglas fir tussock moth, elm spanworm, fall webworm, fruittree leafroller, greenstriped mapleworm, gypsy moth, jack pine budworm, mimosa webworm, pine butterfly, redhumped caterpillar, saddleback caterpillar, saddle prominent caterpillar, spring and fall cankerworm, spruce budworm, tent caterpillar, tortrix, and western tussock moth. Likewise, turf grasses are often attacked by pests such as armyworm, sod webworm, and tropical sod webworm.
Because crops of commercial interest are often the target of insect attack, environmentally-sensitive methods for controlling or eradicating insect infestation are desirable in many instances. This is particularly true for farmers, nurserymen, growers, and commercial and residential areas which seek to control insect populations using eco-friendly compositions.
The most widely used environmentally-sensitive insecticidal formulations developed in recent years have been composed of microbial pesticides derived from the bacterium Bacillus thuringiensis. B. thuringiensis is a Gram-positive bacterium that produces crystal proteins or inclusion bodies which are specifically toxic to certain orders and species of insects. Many different strains of B. thuringiensis have been shown to produce insecticidal crystal proteins. Compositions including B. thuringiensis strains which produce insecticidal proteins have been commercially-available and used as environmentally-acceptable insecticides because they are quite toxic to the specific target insect, but are harmless to plants and other non-targeted organisms.
1.2.1 δ-Endotoxins
δ-endotoxins are used to control a wide range of leaf-eating caterpillars and beetles, as well as mosquitoes. These proteinaceous parasporal crystals, also referred to as insecticidal crystal proteins, crystal proteins, Bt inclusions, crystaline inclusions, inclusion bodies, and Bt toxins, are a large collection of insecticidal proteins produced by B. thuringiensis that are toxic upon ingestion by a susceptible insect host. Over the past decade research on the structure and function of B. thuringiensis toxins has covered all of the major toxin categories, and while these toxins differ in specific structure and function, general similarities in the structure and function are assumed. Based on the accumulated knowledge of B. thuringiensis toxins, a generalized mode of action for B. thuringiensis toxins has been created and includes: ingestion by the insect, solubilization in the insect midgut (a combination stomach and small intestine), resistance to digestive enzymes sometimes with partial digestion actually “activating” the toxin binding to the midgut cells, formation of a pore in the insect cells and the disruption of cellular homeostasis (English and Slatin, 1992).
1.2.2 Genes Encoding Crystal Proteins
Many of the δ-endotoxins are related to various degrees by similarities in their amino acid sequences. Historically, the proteins and the genes which encode them were classified based largely upon their spectrum of insecticidal activity. The review by Höfte and Whiteley (1989) discusses the genes and proteins that were identified in B. thuringiensis prior to 1990, and sets forth the nomenclature and classification scheme which has traditionally been applied to B. thuringiensis genes and proteins: cryI genes encode lepidopteran-toxic CryI proteins. cryII genes encode CryII proteins that are toxic to both lepidopterans and dipterans. cryIII genes encode coleopteran-toxic CryIII proteins, while cryIV genes encode dipteran-toxic CryIV proteins, etc. Based on the degree of sequence similarity, the proteins were further classified into subfamilies; more highly related proteins within each family were assigned divisional letters such as CryIA, CryIB, CryIC, etc. Even more closely related proteins within each division were given names such as CryIC1, CryIC2, etc.
Recently a new nomenclature was developed which systematically classifies the Cry proteins based upon amino acid sequence homology rather than upon insect target specificities. This classification scheme, including most of the known toxins but not including allelic variations in individual polypeptides, is summarized in Table 1.
TABLE 1KNOWN B. THURINGIENSIS δ-ENDOTOXINS,GENBANK ACCESSION NUMBERS,AND REVISED NOMENCLATUREANewOldGenBank Accession #Cry1Aa1CryIA(a)M11250Cry1Aa2CryIA(a)M10917Cry1Aa3CryIA(a)D00348Cry1Aa4CryIA(a)X13535Cry1Aa5CryIA(a)D17518Cry1Aa6CryIA(a)U43605Cry1Ab1CryIA(b)M13898Cry1Ab2CryIA(b)M12661Cry1Ab3CryIA(b)M15271Cry1Ab4CryIA(b)D00117Cry1Ab5CryIA(b)X04698Cry1Ab6CryIA(b)M37263Cry1Ab7CryIA(b)X13233Cry1Ab8CryIA(b)M16463Cry1Ab9CryIA(b)X54939Cry1Ab10CryIA(b)Cry1Ac1CryIA(c)M11068Cry1Ac2CryIA(c)M35524Cry1Ac3CryIA(c)X54159Cry1Ac4CryIA(c)M73249Cry1Ac5CryIA(c)M73248Cry1Ac6CryIA(c)U43606Cry1Ac7CryIA(c)U87793Cry1Ac8CryIA(c)U87397Cry1Ac9CryIA(c)U89872Cry1Ac10CryIA(c)AJ002514Cry1Ad1CryIA(d)M73250Cry1Ae1CryIA(e)M65252Cry1Ba1CryIBX06711Cry1Ba2X95704Cry1Bb1ET5L32020Cry1Bc1CryIb(c)Z46442Cry1Bd1CryE1Cry1Ca1CryICX07518Cry1Ca2CryICX13620Cry1Ca3CryICM73251Cry1Ca4CryICA27642Cry1Ca5CryICX96682Cry1Ca6CryICX96683Cry1Ca7CryICX96684Cry1Cb1CryIC(b)M97880Cry1Da1CryIDX54160Cry1Db1PrtBZ22511Cry1Ea1CryIEX53985Cry1Ea2CryIEX56144Cry1Ea3CryIEM73252Cry1Ea4U94323Cry1Eb1CryIE(b)M73253Cry1Fa1CryIFM63897Cry1Fa2CryIFM63897Cry1Fb1PrtDZ22512Cry1Ga1PrtAZ22510Cry1Ga2CryIMY09326Cry1Gb1CryH2Cry1Ha1PrtCZ22513Cry1Hb1U35780Cry1Ia1CryVX62821Cry1Ia2CryVM98544Cry1Ia3CryVL36338Cry1Ia4CryVL49391Cry1Ia5CryVY08920Cry1Ib1CryVU07642Cry1Ja1ET4L32019Cry1Jb1ET1U31527Cry1Ka1U28801Cry2Aa1CryIIAM31738Cry2Aa2CryIIAM23723Cry2Aa3D86084Cry2Ab1CryIIBM23724Cry2Ab2CryIIBX55416Cry2Ac1CryIICX57252Cry3Aa1CryIIIAM22472Cry3Aa2CryIIIAJ02978Cry3Aa3CryIIIAY00420Cry3Aa4CryIIIAM30503Cry3Aa5CryIIIAM37207Cry3Aa6CryIIIAU10985Cry3Ba1CryIIIBX17123Cry3Ba2CryIIIBA07234Cry3Bb1CryIIIB2M89794Cry3Bb2CryIIIC(b)U31633Cry3Ca1CryIIIDX59797Cry4Aa1CryIVAY00423Cry4Aa2CryIVAD00248Cry4Ba1CryIVBX07423Cry4Ba2CryIVBX07082Cry4Ba3CryIVBM20242Cry4Ba4CryIVBD00247Cry5Aa1CryVA(a)L07025Cry5Ab1CryVA(b)L07026Cry5Ba1PS86Q3U19725Cry6Aa1CryVIAL07022Cry6Ba1CryVIBL07024Cry7Aa1CryIIICM64478Cry7Ab1CryIIICbU04367Cry8Aa1CryIIIEU04364Cry8Ba1CryIIIGU04365Cry8Ca1CryIIIFU04366Cry9Aa1CryIGX58120Cry9Aa2CryIGX58534Cry9Ba1CryIXX75019Cry9Ca1CryIHZ37527Cry9Da1N141D85560Cry10Aa1CryIVCM12662Cry11Aa1CryIVDM31737Cry11Aa2CryIVDM22860Cry11Ba1Jeg80X86902Cry12Aa1CryVBL07027Cry13Aa1CryVCL07023Cry14Aa1CryVDU13955Cry15Aa134 kDaM76442Cry16Aa1cbm71X94146Cry17Aa1cbm71X99478Cry18Aa1CryBP1X99049Cry19Aa1Jeg65Y08920Cry20Aa1U82518Cry21Aa1I32932Cry22Aa1I34547Cyt1Aa1CytAX03182Cyt1Aa2CytAX04338Cyt1Aa3CytAY00135Cyt1Aa4CytAM35968Cyt1Ab1CytMX98793Cyt1Ba1U37196Cyt2Aa1CytBZ14147Cyt2Ba1“CytB”U52043Cyt2Ba2“CytB”AF020789Cyt2Ba3“CytB”AF022884Cyt2Ba4“CytB”AF022885Cyt2Ba5“CytB”AF022886Cyt2Bb1U82519aAdapted from: http://epunix.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/index.html1.2.3 Bioinsecticide Polypeptide Compositions
The utility of bacterial crystal proteins as insecticides was extended beyond lepidopterans and dipteran larvae when the first isolation of a coleopteran-toxic B. thuringiensis strain was reported (Krieg et al., 1983; 1984). This strain (described in U.S. Pat. No. 4,766,203, specifically incorporated herein by reference), designated B. thuringiensis var. tenebrionis, is reported to be toxic to larvae of the coleopteran insects Agelastica alni (blue alder leaf beetle) and Leptinotarsa decemlineata (Colorado potato beetle).
U.S. Pat. No. 5,024,837 also describes hybrid B. thuringiensis var. kurstaki strains which showed activity against lepidopteran insects. U.S. Pat. No. 4,797,279 (corresponding to EP 0221024) discloses a hybrid B. thuringiensis containing a plasmid from B. thuringiensis var. kurstaki encoding a lepidopteran-toxic crystal protein-encoding gene and a plasmid from B. thuringiensis tenebrionis encoding a coleopteran-toxic crystal protein-encoding gene. The hybrid B. thuringiensis strain produces crystal proteins characteristic of those made by both B. thuringiensis kurstaki and B. thuringiensis tenebrionis. U.S. Pat. No. 4,910,016 (corresponding to EP 0303379) discloses a B. thuringiensis isolate identified as B. thuringiensis MT 104 which has insecticidal activity against coleopterans and lepidopterans.
1.2.4 Molecular Genetic Techniques Facilitate Protein Engineering
The revolution in molecular genetics over the past decade has facilitated a logical and orderly approach to engineering proteins with improved properties. Site specific and random mutagenesis methods, the advent of polymerase chain reaction (PCR™) methodologies, and related advances in the field have permitted an extensive collection of tools for changing both amino acid sequence, and underlying genetic sequences for a variety of proteins of commercial, medical, and agricultural interest.
Following the rapid increase in the number and types of crystal proteins which have been identified in the past decade, researchers began to theorize about using such techniques to improve the insecticidal activity of various crystal proteins. In theory, improvements to δ-endotoxins should be possible using the methods available to protein engineers working in the art, and it was logical to assume that it would be possible to isolate improved variants of the wild-type crystal proteins isolated to date. By strengthening one or more of the aforementioned steps in the mode of action of the toxin, improved molecules should provide enhanced activity, and therefore, represent a breakthrough in the field. If specific amino acid residues on the protein are identified to be responsible for a specific step in the mode of action, then these residues can be targeted for mutagenesis to improve performance
1.2.5 Structural Analyses of Crystal Proteins
The combination of structural analyses of B. thuringiensis toxins followed by an investigation of the function of such structures, motifs, and the like has taught that specific regions of crystal protein endotoxins are, in a general way, responsible for particular functions.
Domain 1, for example, from Cry3Bb and Cry1Ac has been found to be responsible for ion channel activity, the initial step in formation of a pore (Walters et al., 1993; Von Tersch et al., 1994). Domains 2 and 3 have been found to be responsible for receptor binding and insecticidal specificity (Aronson et al., 1995; Caramori et al., 1991; Chen et al. 1993; de Maagd et al., 1996; Ge et al., 1991; Lee et al., 1992; Lee et al., 1995; Lu et al., 1994; Smedley and Ellar, 1996; Smith and Ellar, 1994; Rajamohan et al., 1995; Rajamohan et al., 1996; Wu and Dean, 1996). Regions in domain 2 and 3 can also impact the ion channel activity of some toxins (Chen et al., 1993, Wolfersberger et al., 1996; Von Tersch et al., 1994).
1.3 Deficiencies in the Prior Art
Unfortunately, while many laboratories have attempted to make mutated crystal proteins, few have succeeded in making mutated crystal proteins with improved lepidopteran toxicity. In almost all of the examples of genetically-engineered B. thuringiensis toxins in the literature, the biological activity of the mutated crystal protein is no better than that of the wild-type protein, and in many cases, the activity is decreased or destroyed altogether (Almond and Dean, 1993; Aronson et al., 1995; Chen et al., 1993, Chen et al., 1995; Ge et al., 1991; Kwak et al., 1995; Lu et al., 1994; Rajamohan et al., 1995; Rajamohan et al., 1996; Smedley and Ellar, 1996; Smith and Ellar, 1994; Wolfersberger et al., 1996; Wu and Aronson, 1992).
For a crystal protein having approximately 650 amino acids in the sequence of its active toxin, and the possibility of 20 different amino acids at each position in this sequence, the likelihood of arbitrarily creating a successful new structure is remote, even if a general function to a stretch of 250-300 amino acids can be assigned. Indeed, the above prior art with respect to crystal protein gene mutagenesis has been concerned primarily with studying the structure and function of the crystal proteins, using mutagenesis to perturb some step in the mode of action, rather than with engineering improved toxins.
Collectively, the limited successes in the art to develop synthetic toxins with improved insecticidal activity have stifled progress in this area and confounded the search for improved endotoxins or crystal proteins. Rather than following simple and predictable rules, the successful engineering of an improved crystal protein may involve different strategies, depending on the crystal protein being improved and the insect pests being targeted. Thus, the process is highly empirical.
Accordingly, traditional recombinant DNA technology is clearly not routine experimentation for providing improved insecticidal crystal proteins. What are lacking in the prior art are rational methods for producing genetically-engineered B. thuringiensis crystal proteins that have improved insecticidal activity and, in particular, improved toxicity towards a wide range of lepidopteran insect pests.